Field of the Invention
This invention relates generally to malaria and the study of Plasmodium parasites. More particularly it relates to Plasmodium parasites genetically attenuated by gene disruption.
Background Art
Over 40 percent of the world's population is at risk for exposure to malaria. More than 250,000 new clinical malaria cases occur annually resulting in 800,000 to 1.2 million deaths most of which are children in sub-Saharan Africa suffering from a severe P. falciparum infection [1]. Malaria remains a global health crisis and there is a dire need for a highly effective malaria vaccine.
Recent results from the clinical trials of a malaria vaccine using a single recombinant protein as immunogen, RTS,S subunit vaccine with AS01 adjuvant; have shown modest protection. [2]. Although these results are potentially useful to reduce the global health burden of malaria, a whole sporozoite vaccine approach would provide a broader immunogenic spectrum and should be much more potent in conferring protection, thereby forming the next generation of malaria vaccines. Vaccination with live sporozoites is safe when parasite development is halted prior to the pathogenic post-hepatic blood stage. For this end, attenuation of sporozoites to affect pre-pathogenic liver-stage arrest of parasite development can be accomplished with radiation attenuated sporozoites (RAS) [47], genetically attenuated parasites (GAP) [36], and chemically attenuated sporozoites (CAS) [48]. RAS immunization has a long standing track record of proven efficacy in rodents [3], monkeys [4] and man [5, 6, 7, 47]. Indeed, in a recent clinical trial such a vaccine (Sanaria® PfSPZ Vaccine) protected 6 out of 6 human volunteers at the highest dose level and was completely safe [49]. In rodent models, the protective efficacies conferred by most GAP vaccines are similar to RAS. PfΔp52Δp36 is the only GAP vaccine that has been assessed in humans, but the trial in which the GAP Pf sporozoites were administered by mosquito bite had to be terminated because of breakthrough infections in one volunteer during immunization [50]. Both RAS and GAP vaccination strategies rely on the one hand on complete developmental arrest of the attenuated parasite at the liver stage of development in host hepatocytes in order to prevent breakthrough blood infection and the subsequent signs, symptoms and pathology of malaria, and on the other hand, the requisite immune responses that result in protection.
From a product manufacturing perspective there are advantages of a GAP vaccine approach. It is comprised of a parasite population with a homogeneous attenuation etiology. The genetic attenuation is an irreversible, intrinsic characteristic of the parasite and its attenuation is not dependent upon external (e.g. radiation, host drug metabolism) factors. Furthermore, in the GAP manufacturing process operators are never exposed to a Pf parasite that can cause disease. GAPs go into developmental arrest in the hepatocyte at the time point predestined by the specific gene deletion. Most GAPs that have been examined, like Δp52, Δp36, Δuis3, Δuis4 and Δslarp/Δsap1, arrest at early liver stage. Other GAPs, like Δfabb/f arrest in the late liver stage. Despite this apparent abundance of GAP vaccine candidates, it has proved to be very difficult to generate a safe and protective GAP in Plasmodium species of human host range, e.g., P. falciparum. For instance, unequivocal orthologs of the uis3 and uis4 genes in P. berghei and P. yoelii are absent in the P. falciparum genome (www.PlasmoDB.org) and can therefore not be made into a vaccine product. Breakthrough infection has also been a problem. In the P. berghei model liver stage arrest of the Δp52 [13], the Δp52&p36 and the Δfabb/f [14] parasites was not complete in that these mutants were capable of maturing in the liver in low numbers, resulting in a blood stage infection and malaria pathology in mice. Moreover, very low numbers of replicating Δp52&p36 P. falciparum parasites were observed in primary human hepatocyte cultures [14], and a breakthrough infection was observed in a clinical trial of Δp52&p36 P. falciparum parasites [50].
Thus, there remains a need for new Plasmodium GAP candidates that completely arrest in the liver stage (safety) and with which immunization confers an immune response and long-lasting protection (efficacy). Such a GAP candidate is the focus of this application.